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首页> 外文期刊>The protein journal >Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia
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Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia

机译：贾第鞭毛虫重组NADH氧化酶的克隆，表达及鉴定

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摘要

The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O-2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1): 155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.
机译：NADH氧化酶家族的酶可通过将分子O-2还原为H2O2，H2O或两者同时催化NADH的氧化。在原生动物寄生虫贾第鞭毛虫中，NADH氧化酶（GlNOX）产生H2O作为终产物，而没有产生H2O2。 GlNOX与寄生虫的新陈代谢，细胞内的氧化还原调节以及对目前用于抗贾第鞭毛虫病的药物的抗性有关。因此，从不同的角度来看，它是一种有趣的蛋白质。在这项工作中，GlNOX基因从基因组兰伯氏菌DNA中扩增出，并在大肠杆菌中以His-Tagged蛋白的形式表达。然后，通过固定的金属亲和层析纯化该酶，进行表征，并将其性质与先前从滋养体中分离的内源酶的性质进行比较（Brown等人在Eur J Biochem 241（1）：155-161，1996）。与稀缺和不稳定的滋养体提取酶相比，重组异源表达系统和一步纯化方法可生产出稳定，高产，高纯度的蛋白质。重组酶主要类似于内源蛋白。如果发现差异，则归因于方法上的差异或工件。这种均质，纯净和功能性的蛋白质制剂可用于GlNOX的详细结构或功能研究，这将提供对G.lamblia的生物学和致病性的更深入了解。

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